Some standard content:
ICS13.300;13.020.40
National Standard of the People's Republic of China
GB/T21858—2008
Chemicals
Bioconcentration
Semi-static fish test
Chemicals-Bioconcentration-Semi-static fish test Issued on 2008-05-12
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China Administration of Standardization of the People's Republic of China
Implementation on 2008-09-01
GB/T21858--2008
1 Scope
Terms and Definitions
Information on Test Articles
Overview of Methods
Instruments and Equipment
Test Preparation
Test Procedure||tt ||Quality Assurance and Quality Control
Data and Report
Appendix A (Informative Appendix)
Appendix B (Informative Appendix)
Appendix C (Informative Appendix)
Appendix D (Informative Appendix)
References
Chemical Properties of Qualified Dilution Water
Recommended Test Fish
Prediction of Duration of Absorption and Removal Phases1gP. Theoretical Sample Sampling for Bioconcentration Test of Test Substance with w=4Calculation of Bioconcentration Factor
GB/T 21858--2008
This standard is equivalent to the Organization for Economic Cooperation and Development (OECD) Chemical Testing Guide No. 305 (1996) "Bioconcentration: Flow-through Fish Test (English Version)".
This standard has been edited as follows:
Change the measurement unit to the legal measurement unit of my country. Appendices A, B, C, D and E of this standard are informative appendices. This standard was proposed and managed by the National Technical Committee for Standardization of Hazardous Chemicals Management (SAC/TC251). The responsible drafting unit of this standard is: Chemical Registration Center of the Ministry of Environmental Protection. The participating drafting units of this standard are: Shanghai Testing Center, Shanghai Academy of Environmental Sciences, Nanjing Institute of Environmental Sciences of the Ministry of Environmental Protection. The main drafters of this standard are: Mao Yan, Zhou Hong, Yu Xiangyi, Zhao Huaqing, Yin Haowen, Shen Genxiang, Liu Jining, ate.net
1 Scope
Semi-static fish test for bioaccumulation of chemicals GB/T 21858-2008
This standard specifies the method overview, test preparation, test procedures, quality assurance and quality control, data and report of the flow-through fish test for bioaccumulation of chemicals.
This standard is applicable to testing and evaluation. The value of the bioaccumulation of stable organic chemicals between 1.5 and 6.0 is also applicable to the testing and evaluation of the bioaccumulation of highly lipophilic organic chemicals with P>6.0. 2 Terms and Definitions
The following terms and definitions apply to this standard. 2.1
Bioconcentration bioconcentration/bioaccumulation The increase in the concentration of a test substance in the test organism (or specific tissue) relative to the concentration of the substance in the test medium. 2.2
Bioconcentration factor bioconcentration factor, Bc The ratio of the concentration of a test substance in the test organism (or specific tissue) to the concentration of the substance in the test medium at any time during the absorption phase of the test. BCF can also be directly calculated from the kinetic equation rate constant (K1/K2), recorded as BCFE. 2.3
Steady-state Plateal or steady-state The state when the curve of the concentration of the test substance in the fish body against time tends to be parallel to the time axis (the change range is ±20%). 2. 4
Steady state bioconcentration factor, BCFaThe ratio of the concentration of a test substance in the test organism (or specific tissue) to the concentration of the substance in the test medium under steady state. 2. 5
Octanol-water partition coefficient, PawThe ratio of the concentration of a substance when it reaches equilibrium in the two-phase medium of octanol-water: usually expressed as P. 2.6
Absorption rate constant, KThe rate of increase of the concentration of the test substance in the fish body or its specific tissue when the test fish is exposed to the medium containing the test substance. Usually expressed as d-1.
Depuration (loss) rate constant, K2The rate at which the concentration of the test substance in the fish body or its specific tissue decreases after the test fish is transferred from the medium containing the test substance to the medium without the test substance. Usually expressed as d-1. 3 Information on test substances
Solubility in water;
Partition coefficient between n-octanol and water (P);
Hydrolyzability;
Determination of the phototransformation of chemicals in water under sunlight or simulated sunlight and under the illumination conditions of bioconcentration tests; Surface tension (if there is no P);
GB/T21858—2008
Vapor pressure;
Results of rapid biodegradability tests:
Toxicity of the test substances to the test fish, such as LCso value; +
Appropriate analytical method (with known accuracy, precision and sensitivity); Details of sample preparation and storage;
Analytical detection limits of the test substances in water and fish; When the test substance is labeled with C, the percentage of impurities carried by radioactive substances should be understood. 4 Overview of the Method
4.1 Principle
The bioconcentration test consists of two stages: the exposure (absorption phase and post-exposure elimination) phase. In the absorption phase, the test fish are exposed to two or more concentrations of the test substance in aqueous solution. The absorption phase is generally 28 days, unless it can be shown that the equations in C can be used to predict the duration of the absorption phase and the time to steady state. If absorption is demonstrated to have been achieved before this time, the absorption phase is extended until the steady state phase is reached, with a maximum of 60 days. The test medium is replaced regularly.
The elimination phase begins. The elimination phase is not necessary until the absorption reaches a steady state and the absorption of the test substance is reduced, unless a model of the bioconcentration factor (BCF) is established using a virtual blank control (see Appendix E for the calculation of the BCF using the measured total wet weight of fish flesh) during the absorption phase. When the value is normal
(viscera), special
test organs (devouring
test substances in the test fish
4.2 Reference substances
No reference substances are recommended.
5 Instruments and equipment
Dissolved oxygen meter;
Water meter;
pH meter;
Analytical balance;
TOC Analyzer;
Water quality measuring instrument;
At the same time,
Aquarium tools made of chemically inert materials! Temperature control equipment;
Laboratory common glassware,
Test substance analysis instrument;
Sample pretreatment instruments and equipment.
6 Experimental preparation
6.1 Test fish
Number model. If
Fish body fat
The amount of. Except for the two groups of different concentrations of the test substance, the more complex absorption rate constant, the clearance rate constant and the edible part (fish meat) and the river should be selected. For non-edible part test substances, the test size should be P>3). In the determination of the test substance, the test species should be selected according to the following criteria: easy to obtain, suitable size and easy to domesticate in the laboratory, as well as entertainment, commercial, ecological value, sensitivity and successful selection precedents. The recommended test species in GB/T21858-2008 are shown in Appendix B. Other fish species can also be used, but the test conditions should be adjusted accordingly, and the reasons for the selection of species and the test methods should be stated in the report. 6. 1.2 Fish egg culture
The test fish should be acclimated in water at the same temperature as the test for more than 2 weeks, and feed a feed equivalent to 1% to 2% of the fish body weight every day. The fat and total protein content of the feed should be measured. It is recommended to measure the content of pesticides and heavy metal chips. Within 48 hours of the start of acclimation of the test fish, record their mortality and judge according to the following breeding standards: a) The mortality rate of the test fish within 7 days is greater than 10%: discard the entire batch of fish; b) The mortality rate of the test fish within 7 days is between 5% and 10%: raise for another day; e) The mortality rate of the test fish within 7 days is less than 5%: accept the current batch of fish. If the mortality rate of the test fish exceeds 5% within the second 7 days, discard the entire batch of fish
Deformed or diseased fish cannot be used as test fish and should be discarded. No disease treatment should be given to the fish during the test and two weeks before the test. If adult fish are used in the test, it should be reported whether the test fish is male, female or a mixture of both. If male and female fish are omitted in the test, there should be no significant difference in the fat content of the missing fish. Male and female fish should be kept together. 6.2 Water for culture
Water for culture should generally be unpolluted and of the same quality as natural water. The test fish species should be able to survive, grow and reproduce in the culture water and should not show any abnormal appearance or behavior during the culture period. The water characteristics should include at least pH, hardness, particulate matter, total organic carbon, ammonium and nitrite content, magnetic density and salinity (for marine fish only). Appendix A recommends the maximum concentration values of some parameters of water and seawater.
7 Test Procedure
7.1 Preparation
7.1.1 Test Container
The test container should be made of chemically inert materials and have no significant adsorption to the test substance. It is recommended to use Teflon materials, stainless steel or glass tubes, and keep plastic hoses to a minimum. For substances with high adsorption coefficients, silanized glass is required. Used containers must be discarded.
7.1.2 Test dilution water
Source of dilution water and water quality of homotrophic water.
During the entire test period, the quality of the test water should remain stable, the pH value should be between 6.0 and 8.5, and the pH value should be kept within the range of ±0.5. The dilution water should be sampled and analyzed regularly, including the determination of heavy metals (such as Cu, Pb, Za, Hg, Cd, Ni), major anions and cations (such as Ca2+, Mg+, Na, K+, CI-, SO42-), pesticides (such as total organic phosphorus and total organochlorine pesticides), total organic carbon and suspended solids. The maximum acceptable mass concentration of natural particulate matter in the dilution water is 5 mg/L (intercepted dry matter of 0.45μm filter membrane), and the maximum acceptable value of total organic carbon is 2Ⅱ/L (see Appendix A). During the entire test, the organic carbon from other sources in the test container should not exceed the organic carbon from the test substance. If an adjuvant is used, it should not exceed 10 mg/(±20%). 7.1.3 Preparation of test substance solution
Prepare a stock solution of the test substance of appropriate concentration. It is recommended to prepare the stock solution by simply mixing or stirring the test substance in the dilution water. Cosolvents or dispersants should be avoided or used with caution as much as possible. The cosolvents that can be used are ethanol, methanol, ethylene glycol monomethyl ether, ethylene glycol dimethyl ether, dimethylformamide and triethylene glycol. The dispersants that can be used are Crenophor RII4o (ethoxylated hydrogenated sesame), Tween 80 (dehydrated sorbitan monooleate polyoxyethylene ether), 0.01% methylcellulose and HCO-40 (ethoxylated hydrogenated sesame oil). 7.2 Experimental operation
7.2.1 Preliminary test
Optimize the experimental conditions, such as the selection of test substance concentration, the duration of absorption phase and elimination phase:3
GB/T21858—2008
7.2.2 Experimental design
Fish should be exposed to at least two concentrations of the test substance in aqueous solution. The higher (or highest) concentration should be 1% of the acute LCs, and the concentration should be more than 10 times the detection limit of the analytical method for the test substance in water. The highest test concentration can also be estimated by dividing the 96h acute LC60 by the appropriate acute-chronic ratio. If possible, other concentration series can also be selected. If the upper concentration cannot be prepared due to the limitation of the Cso of 1 and the lower limit of analysis, a concentration step of less than 10 times can be used, or the use of 14C-labeled test substances can be considered. In any case, concentrations greater than the solubility of the test substance shall not be used. When a cosolvent is used in the test, its volume concentration shall be less than 0.1 μg/L and the concentration shall be the same in all test containers. The organic carbon content of the cosolvent shall be determined.
A dilution water control group or a cosolvent control group shall be established. 7.2.3 Conditions
7.2.3.1 Duration of the absorption phase
The duration of the absorption phase shall be obtained by practical experience or by means of a certain empirical relationship, such as the water solubility of the test substance or the partition coefficient of n-octanol-water (Table C). The duration of the absorption phase is a stable state, which should be a long absorption phase. 7.2.3.2 Clearance phase The duration of the clearance phase is too long, for example, the time required for the absorption phase exceeds the sample absorption limit. If the time required for the absorption phase is too long, it may exceed the sample absorption limit. Use one time to judge the test substance!
The chemical substance at a constant state concentration
is analyzed under a certain condition
. The activity of the test substance in the fish
7.2.3.3 Test medium
During the test, when the test medium is not filled
, the medium should be quickly diluted
to ensure that the test substance and the dilute
7.2.3.4 The
of the test fish is balanced by the original test
|. t|| Gong with the tail
Hua (see attached
new doubling time (if the super
contains fresh test
can be balanced, such as on the 28th day, there is still a shorter time to reach stability.
explanation). If it reaches 95% of the removal amount, then the time of the removal phase can be shortened (for example, the rate constant. Please note that the removal period time should depend on the frequency of changing the test medium. Do not change the tester. Fresh test material should be prepared in advance, at least 4 If a larger statistical sample is required, more fish tails will be needed. When testing, the weight group of test fish should be selected, and the weight of the smallest fish should not be less than two-thirds of the largest fish. The weight and age of all test fish should be recorded. It is recommended to weigh more test fish before the test. They should be of the same age and from the same source. 』wwW.bzxz.Net
7.2. 3.5 Loading capacity
The recommended loading rate is fish weight (weight) 0.1 g/(eg/(d . L)
soil within 20%, and the dissolved oxygen concentration is not less than 60% of the saturation value, the carrying capacity can be increased. When selecting the appropriate carrying capacity, the normal living environment required by the fish species should be considered. 7.2.3.6 Feeding
If the concentration fluctuation of the test substance can be maintained
during the test period, use the same feed as during the other breeding periods. Feed the fish daily with a feed equivalent to 1% to 2% of the body weight of the fish. It is recommended to sample and weigh the test fish before the test. The weight of the fish in each test can be estimated based on the weight of the most recently sampled fish. The weight of the fish in the test should not be weighed.
Within 30min to 60min after feeding each day, use a siphon to remove the remaining bait and fish excrement in the test tank. Keep the test tank as clean as possible throughout the test period to ensure that the concentration of organic matter is as low as possible. If the test medium is changed, feed it 1h to 2h before each change. 7.2. 3.7 Light and temperature
During the test, the light adjustment period should be 12h~16h, and the temperature should be controlled within ±2℃ of the most suitable temperature for the test fish species (see Appendix B). 4
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GB/T21858—2008
7,2.4 Frequency of water quality measurement
During the test, the dissolved oxygen, TOC, pH and temperature in all test containers should be measured. The total hardness and salinity should be measured in the control group and the higher or highest test concentration group. In the absorption stage, the dissolved oxygen should be measured at least 3 times, at the beginning of the test, in the middle of the absorption stage and at the end. Once a week in the clearance stage. TOC should be measured before the test fish are introduced (24h~48h before the absorption stage), at least once a week in the absorption stage and the clearance stage. H should be measured at the beginning and end of each stage. Hardness should be measured once for each test, and temperature should be measured daily, and the temperature in at least one test container should be continuously monitored. 7.2.5 Sampling and analysis of test fish and water
7.2.5.1 Sampling schedule for test fish and water
Water samples should be collected from the test tank for test substance concentration determination before adding test fish, during the absorption phase, and during the clean-up phase. The frequency of water sampling should be no less than that of fish sampling and should be carried out before feeding. Fish samples should be collected at least 5 times during the absorption phase and at least 4 times during the clean-up phase. In some cases, it is difficult to calculate two sufficiently accurate BCF values with only a few samples, so it is recommended to increase the number of samplings in both phases (see Appendix D). Appendix D gives an example of a sampling schedule. The relevant schedule can also be quickly derived by using other estimated values of P to calculate the exposure time for 95% absorption.
Sampling should be continued during the absorption phase until steady state occurs or 28 days of the test period (whichever is shorter). If steady state has not been reached within 28 days, sampling should continue until steady state is reached or 60 days (whichever is shorter). Before the start of the clean-up phase, the test fish should be transferred to a container of fresh water.
7.2.5.2 Sampling and sample preparation
Water samples for analysis and determination are collected from the middle area of the test tank using an inert material tube according to the siphon principle. The container should be kept as clean as possible and the total organic carbon content should be tested during the absorption and removal stages. Each time fish samples are taken, an appropriate number of test fish (at least 4) are taken from the test tank, the test fish are quickly rinsed with water, the surface water is sucked dry and the fish are quickly killed in the most humane way and weighed. Fish and water samples should be measured as soon as possible after collection to avoid degradation or other losses, and the absorption and removal rates are approximately calculated. If samples cannot be measured in time, appropriate methods should be used to preserve the samples. The method of preserving the specific test substance, storage time and extraction method should be mastered before the start of the test.
7.2.5.3 Analytical methods
For specific test substances, it is necessary to check the accuracy and reproducibility of the analytical method, as well as the recovery rate of the test substance in water and fish. In addition, the test substance should not be detected in the dilution water. If necessary, the C and C values measured in the test should be corrected. 7.2.5.4 Determination of fish samples
When radioactive labeled substances are used in the test, the total amount of radioactive labeled substances (including parent and metabolites) can be determined, or the parent test substance can be determined separately after washing the sample. The main metabolite can also be identified at the steady state or at the end of the absorption phase (whichever is shorter). If the BCF value obtained based on the total amount of radioactive labeled residues is not less than 1000, it is necessary to identify whether the degradation rate of the total residue of the test substance in the fish tissue at steady state is not less than 10%, especially for special categories of compounds such as pesticides. If the degradation rate of the total residue of the test substance in the fish tissue is not less than 10%, it is necessary to determine the degradation of the test substance in water. The content of the test substance in each fish should usually be determined. If this is not possible, the test fish with the same concentration should be combined after each sampling, but this will not be able to perform mathematical and statistical analysis. If mathematical statistics are really needed, the sample yield (variable test tail number) should be fully considered in the experimental design.
BCF value can be expressed as a function of the total wet weight of the test fish, and for highly lipophilic substances, it can also be expressed as a function of the fat content of the fish. If possible, the fat content of the fish sample should be measured every time it is sampled. The methane/methanol extraction method is recommended as the standard method. The results obtained by different test methods will not be exactly the same, so the detailed steps of the method used should be given. If possible, the same sample extract should be used for fat determination and test substance determination, because the chromatographic analysis of the test substance determination usually requires the removal of fat. The difference between the fat content (expressed in mg/kg wet weight) of the test fish at the end of the test and at the beginning of the test should be less than or equal to ±25%, and the dry weight of the fish tissue should be reported to understand the ratio of fat content converted from wet weight to dry weight.
GB/T 21858—2008
8 Quality Assurance and Quality Control
An effective test shall meet the following conditions;
a) Temperature fluctuation is less than ±2℃;
b) Dissolved oxygen concentration is not less than 60% of the air saturation value; The photoconversion of the test substance under the experimental lighting conditions is controlled, and ultraviolet radiation with a wavelength less than 290nm is filtered out to avoid the test fish from being exposed to abnormal photochemical products;
d) During the absorption period, the concentration of the test substance in the test container is maintained within ±20% of the measured average value; Until the end of the test, the mortality rate or other adverse effects or diseases of the control and test group fish are less than 10%. When the test is extended for several weeks or months, the mortality or other adverse effects of the test fish in the two groups should be less than 5% per month, and shall not exceed 30% during the individual process.
9 Data and Report
Data Processing
Plot the concentration of the test substance in the fish (or specific tissue of the fish) against time on a coordinate paper to form a curve. If the curve has reached a steady state, that is, it has become an approximate asymptote for the time axis, use formula (1) to calculate the bioconcentration factor (BCF6) of the steady state:
Wu Zhong:
C—average concentration of the test substance in the fish tissue: Cw—average concentration of the test substance in the test. When the steady state is not reached, the BCF value of 80% or 95% of the steady state can also be calculated. 1
The kinetic enrichment factor (BCF) can be determined by the ratio of the absorption rate constant of the first exponential sound path to the cumulative removal rate constant. The clearance rate band number K is determined by the clearance curve (i.e., the curve of the concentration of the substance in the fish body against time drawn on the graph), and the absorption rate constant K1 can be calculated based on the given K2 value and the C value from the absorption curve, see Appendix E. A better way to calculate BCFk and K1, K2 is to apply nonlinear parameter estimation software, otherwise the K1, K2 values can be calculated by graphical method. If the clearance curve is obviously not a first-order exponential equation, more complex methods (see Appendix C) and biostatistical methods should be used. 9.2 Test report
The test report must include the following information.
a) Test substance:
Physical properties and related physicochemical properties; -Chemical property data, including organic carbon content: If radioactive labeled substances are used, the exact position of the labeled atoms and the percentage of relevant radioactive impurities. b) Test organisms:
Scientific name, strain, source, pretreatment, domestication, age and individual size, etc. Test conditions:
Test procedure used;
Type, characteristics and photoperiod of light source used; -Test design, such as size and number of test tanks, test medium replacement frequency, number of parallels, number of test fish per parallel, series of test substance concentrations, duration of absorption and clearance periods, frequency of fish and water sampling; method of preparing test stock and frequency of stock replacement (when solvents are used, their concentration, contribution to the organic carbon content of the test solution):
http://GB/T 21858—2008
Established test concentration, mean value of concentration measured in the test vessel and its standard deviation, analytical method: source of dilution water, description of pretreatment, performance of test fish in water and water quality characteristics: pH, hardness, temperature, dissolved oxygen concentration, residual chlorine level (if measured), total organic carbon, suspended particulate matter, salinity of the test medium and any other measured results;
Water quality in the test vessel, pH, hardness, TOC, temperature, dissolved oxygen; detailed information on feeding, such as bait type, source, composition (including fat and protein content at least), feeding amount and frequency; information on the handling of fish and water samples, including detailed preparation, storage, extraction, analytical procedures and precision for the test substance and lipid content.
d) Results:
Results obtained from the preliminary test:
Mortalities of the control group and the exposed groups, abnormal behaviors of the fish observed; -Fat content of the fish;
Curve (including all test data) showing the status of the test substance in the fish until the steady-state absorption and elimination stages; -C and C values for all sampling times (including standard deviation and range of variation), C value of the control group and background value (Cr is expressed in mg/g whole fish wet weight or special fish tissue such as fat wet weight or mg/kg, Cw is expressed in mg/g or mg/kg);
-Steady-state bioconcentration factor (BCF) and kinetic enrichment factor (BCFk), if possible,Absorption and elimination rate constants with 95% confidence limits (expressed in terms of the relevant whole fish, total fat content of the fish body or its special tissues), confidence limits and standard deviations, as well as calculation and data analysis methods for the concentrations of each test substance; when radioactive markers are used, any metabolites measured, if necessary; any abnormal circumstances in the test, adjustments to the test procedures and other relevant information: - Other matters for explanation.
e) Results discussion:
The preliminary test results of the determination method study should avoid results such as "not detected under the method detection limit" as much as possible, because this will not be used to calculate the rate constant.
GB/T 21858--2008
Appendix A
(Informative Appendix)
Chemical Properties of Qualified Dilution Water
Chemical Properties of Qualified Dilution Water See Table A.1. Table A.1 Chemical Properties of Qualified Dilution Water Properties
Particulate Matter
Total Organic Carbon
Free Ammonia
Total Organic Ammonia
Total Organic Ammonia and Polyamine Combined
Total Organic Chlorine
Limit
5 mg/L
2 mg/L
1 μg/L
50 ng/L
50ng/L
25 ng/L
1 μg/L
1 ag/L
1 μ/L
10 ng/L
100 ang/L
100 ng/L
Recommended test fish are shown in Table B.1.
Test fish
Brachydania rerio
Rare Gooeypnsrarus）
Swordtail fish (Xiphoporus helleri)
Blackhead softmouth (Pimephales)
Carp color (Gyprinusarpia
Blue (Oryzias latip's)
Guppy (Poecitia ret
Bluegill sunfish (Li
Rainbow salmon (O
Three-spined stickleback (Gaster
The above list
or the source of parasites.
aehis)
husmykiss
eatus)
Easy to keep and vinegar
They can
Appendix B
(Informative Appendix)
Recommended test fish
Recommended test fish
Test temperature range/℃
20 ~ 25
-21-25
21~25*
GB/T 21858-2008
Test fish body length/cm
3. 0±0.5
3. 0±0. 5
5. 0±2. 0
fish species may be difficult to collect. In controlling diseases, it is important to know the health of the fish and its
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